chapter 10 molec

What is the fundamental requirement of RNA processing?

RNA processing encompasses a variety of modifications that occur to RNA molecules after they are transcribed. These modifications are essential for producing functional RNAs and include:

• Removal of ends from larger precursor RNAs by ribonucleases (RNA-cleaving enzymes).

• RNA splicing

• 5' capping

• Polyadenylation

• RNA editing

• RNA degradation

Name three benefits associated with RNA processing.

RNA processing contributes to:

• regulation of gene expression1

• generation of diverse RNAs from one gene1

• serving as a quality control step1

What are introns and exons?

• Introns are non-coding sequences that interrupt the coding sequence of many eukaryotic genes1

• Exons are the coding segments of a gene1 . Introns are removed during RNA processing, and exons are joined together

What is RNA splicing?

the process of removing introns from a precursor RNA and rejoining the remaining exons to create a functional RNA molecule

What is 5' capping?

modification of eukaryotic pre-mRNAs where the 5' end is modified with a 7-methyl guanosine

What is polyadenylation?

the addition of a long stretch of adenosine nucleotides to the 3' end of eukaryotic pre-mRNAs

RNA editing

involves the insertion, deletion, or chemical modification of bases in an RNA molecule

What are ribonucleases?

RNA-cleaving enzymes that remove the ends of transcripts from larger precursor RNAs

What are the functions of the 5' cap in eukaryotic mRNAs?

The 5' cap is essential for:

• Efficient elongation and termination of transcription2 .

• Subsequent processing of the mRNA2 .

• Acting as a binding site for nuclear export proteins2 .

• Directing the initiation of protein synthesis2 .

• Protecting the 5' end from degradation by exonucleases2

What are the functions of the 3' poly(A) tail in eukaryotic mRNAs?

• Protects the mRNA from degradation by exonucleases3 .

• Plays a role in the initiation of protein synthesis

What are snoRNAs and what is their primary function?

short RNA molecules that guide enzymes to specific sites in rRNA (and some tRNAs) to carry out methylation and pseudouridylation.

What are snRNPs and what role do they play in RNA processing?

complexes of snRNAs and proteins that are key components of the spliceosome and are involved in mRNA splicing

What is the spliceosome?

a large ribonucleoprotein machine composed of snRNPs and many other proteins that carries out the splicing of eukaryotic mRNAs

What are self-splicing introns? Name two groups.

Self-splicing introns are introns that can catalyze their own removal from RNA. Two groups are Group I introns and Group II introns.

What are splice sites?

the locations on a pre-mRNA where introns are removed and exons are joined together

What is alternative splicing?

a process in which different combinations of exons within a gene are spliced together to produce multiple different mature mRNAs

What are splicing enhancers and silencers?

RNA elements that positively affect splicing by increasing the ability of spliceosomes to identify nearby splice sites

silencers (ISSs and ESSs) have the opposite effect, often masking splice sites or blocking spliceosome assembly

What is RNA degradation?

the process by which RNA molecules are broken down into individual nucleotides

How does the 5' cap affect RNA stability in eukaryotes?

5' cap protects the 5' end of eukaryotic mRNAs from degradation by 5' to 3' exonucleases, thus increasing RNA stability

How does the poly(A) tail generally affect RNA stability in eukaryotes?

In eukaryotes, the poly(A) tail generally increases the stability of RNA transcripts

What is deadenylation?

the shortening of the poly(A) tail of eukaryotic mRNAs and is often the first step in their degradation

What is the general role of modifications in tRNAs and rRNAs?

Modifications often appear to be required for the optimal survival and growth of organisms. They can contribute to structural stability and interaction with other molecules

What is the potential function of pseudouridine in rRNA?

the extra hydrogen-bonding capacity of pseudouridine relative to uridine could help stabilize the structure of the ribosome

What type of enzymes catalyze tRNA and some rRNA modifications in bacteria?

In bacteria, modifications are added by site-specific enzymes that catalyze a given modification at a specific site in the RNA

What are pseudouridine synthases and methylases?

pseudouridine synthases catalyze the conversion of uridine to pseudouridine, and methylases add methyl groups to a target molecule

What is the main function of snoRNAs in archaea and eukaryotes?

guide enzymes to the site in the RNA that is to be modified, primarily for ribose methylation and pseudouridylation of rRNAs (and some tRNAs)

What is a snoRNP?

snoRNP is a ribonucleoprotein complex formed by a snoRNA associated with a specific set of proteins, including the modifying enzyme

Name the two major classes of snoRNAs and the modifications they direct.

The two major classes of snoRNAs are:

• Box C/D snoRNAs: direct ribose methylation6 .

• Box H/ACA snoRNAs: direct pseudouridylation

How do snoRNAs exert their effects?

the snoRNA within the snoRNP base-pairs with the target RNA, directing the modification enzymes to particular positions

Describe the structure of the 5' cap.

5' cap is a special 7-methylguanine nucleotide attached to the end of the mRNA via a unique 5'-5' triphosphate linkage

What are the functions of the 5' cap in eukaryotic mRNAs?

The 5' cap is essential for:

• Efficient elongation and termination of transcription.

• Subsequent processing of the mRNA.

• Acting as a binding site for proteins that export the mRNA from the nucleus.

• Directing the initiation of protein synthesis.

• Protecting the 5' end of the mRNA from degradation by 5' to 3' exonucleases

Describe the three steps of 5' cap addition in eukaryotes.

• RNA 5' triphosphatase removes one phosphate from the 5' end.

• Guanyl transferase attaches a GMP molecule via a 5'-5' triphosphate linkage.

• Guanine-7-methyltransferase methylates the transferred guanine

Where and when are eukaryotic mRNAs polyadenylated?

the 3' ends of most eukaryotic mRNAs are modified by the addition of a poly(A) tail as part of the transcription termination process

What are the functions of the 3' poly(A) tail in eukaryotic mRNAs?

• Protects the mRNA from degradation by exonucleases.

• Plays a role in the initiation of protein synthesis

What are alternative polyadenylation sites (APAs)?

multiple sites on some mRNAs where the precursor mRNA can be cleaved and the poly(A) tail added. APAs contribute to the regulation of protein synthesis and expand the range of protein products.

Describe the general process of polyadenylation in eukaryotes.

begins with cleavage of the mRNA, typically after a CA nucleotide pair downstream of an AAUAAA hexamer and upstream of a U- or GU-rich region. Following cleavage, a tail of approximately 200 adenosines is added by poly(A) polymerase

What is the general role of polyadenylation in bacteria?

polyadenylation is generally associated with mRNA destabilization

how are capping and polyadenylation coupled to each other and to transcription?

Capping is required for efficient elongation by RNA polymerase II, and polyadenylation is required for efficient termination. RNA polymerase II also recruits proteins that remove introns

Which domain of RNA polymerase II is responsible for orchestrating mRNA processing events?

The C-terminal domain (CTD) of RPB1, the largest subunit of RNA polymerase II, orchestrates capping, splicing, and polyadenylation

How are mRNA export and translation linked to transcription and processing?

Protein factors required for nuclear export are loaded during transcription, but export-competent complexes are released only after signals from polyadenylation enzymes

What are mRNA localization elements and where are they typically found?

sequences, generally found at the 3' end of the transcript, that allow mRNAs to be transported to specific regions of the cytoplasm for translation

What is the primary role of RNA splicing?

to remove introns from precursor RNAs to produce functional molecules

How are most introns removed from precursor RNAs?

through a series of transesterification reactions

What is exon shuffling and what is its significance?

process where DNA encoding exons can be exchanged and reordered through genetic recombination between intronic DNA. It is thought to have played a major part in the evolution of eukaryotic genomes

How does RNA splicing occur chemically?

occurs by two transesterification reactions. A phosphodiester bond is broken and replaced with an energetically equivalent one

What is the energy requirement for transesterification reactions in splicing?

occur without the net input of energy from ATP because one phosphodiester bond is replaced by another

What are self-splicing introns?

introns that can catalyze their own removal from RNA

Describe the splicing mechanism of Group I introns.

Group I intron splicing involves two sequential transesterifications. The first uses a free guanosine nucleotide to attack the 5' splice site, detaching the intron from exon 1. The second uses the released 3' end of exon 1 to attack the intron-exon 2 junction, joining the exons and releasing the intron

How are splice sites defined in Group I introns?

defined by the three-dimensional structure of the intron and by the recognition of a conserved G-U wobble pairing interaction

Describe the splicing mechanism of Group II introns.

two transesterification reactions where the 2' OH of a specific adenosine within the intron acts as the initial attacking nucleophile, forming a lariat intermediate

how are splice sites defined in Group II introns?

three-dimensional structure of the intron and by specific base-pairings and tertiary interactions

How does the spliceosome identify splice sites?

by recognizing short sequence motifs found in each pre-mRNA

Describe the initial steps of spliceosome assembly.

First, the 5' splice site is recognized by the U1 snRNP. The branch-point sequence is recognized by the branch-point-binding protein (BBP), and the polypyrimidine tract and 3' splice site are bound by components of U2AF

What is the exon junction complex (EJC)?

a set of proteins left behind at splice junctions after splicing, marking the transcript as processed and ready for export and translation. EJCs are involved in quality control during translation

What is trans splicing?

an unusual form of splicing where exons from two separate RNA molecules are joined together

What is the role of the C-terminal tail of RNA polymerase in splicing?

The C-terminal tail of RNA polymerase interacts with splicing components like the U1 snRNP, facilitating co-transcriptional splicing, where splice sites can 'see' each other as they emerge

What are SR proteins and what is their general role in splicing?

(serine/arginine repeats) bind to pre-mRNA, particularly to ESE sequences, and are thought to help recruit other spliceosome components to the splice sites, playing a role in exon definition

What is PTB and what is its role in splicing regulation?

a silencer protein that binds to intronic or exonic elements to silence weak exons, sometimes by competing with U2AF for binding to the polypyrimidine tract or by interfering with spliceosome assembly

What are AU-rich elements (AREs) and how do they affect mRNA stability in eukaryotes?

found in the 3' UTRs of some mRNAs cause instability by directing poly(A) tail removal

Describe the initiation of RNA degradation in many E. coli RNAs

Degradation is often initiated by an endonuclease, most commonly RNase E, which is part of the degradosome

What is the degradosome?

protein complex in E. coli that includes RNase E (an endonuclease), an RNA helicase, and the 3' to 5' exonuclease polynucleotide phosphorylase (PNPase), likely facilitating RNA degradation

What are the two main mRNA decay pathways in eukaryotes after deadenylation?

• Decapping by the DCP2 complex, followed by 5' to 3' degradation by the exonuclease XRN1.

• 3' to 5' degradation by the exosome, a 3' to 5' exonuclease.

Describe how iron levels regulate the stability of transferrin receptor mRNA.

When iron levels are low, iron regulatory proteins (IRP1 and IRP2) bind to iron response elements (IREs) in the 3' UTR of the transferrin receptor mRNA, protecting it from degradation. When iron is high, IRPs do not bind, and the mRNA is degraded

What is RNA interference (RNAi) and in what organisms is it found?

defense mechanism in eukaryotic cells that detects and destroys foreign, often double-stranded RNAs

What are some types of defective mRNAs that cells degrade?

premature translation termination codon, those that lack a termination codon, and those with sequences that can promote ribosome stalling

Name three mRNA decay processes in eukaryotes that target defective mRNAs.

nonsense-mediated mRNA decay (NMD), non-stop decay (NSD), and no-go decay (NGD)

What is tmRNA and what is its function in bacteria?

unique bacterial RNA that functions as both a tRNA and mRNA. It allows ribosomes stalled on defective mRNAs (especially those lacking stop codons) to finish translation using its own coding sequence and promotes the degradation of the defective mRNA